Oxidative modification of proteins has been implicated in the process of aging and a number of pathological disorders. These modifications result in the formation of carbonyl derivatives of amino acid side chains and to protein fragmentation. It has been proposed that some proteins undergo changes in hydrophobicity during aging. The present study was undertaken to determine: (a) if the oxidation of proteins in vitro can lead to an increase in hydrophobicity, and (b) if there is an increase in the hydrophobicity of proteins during aging. It was established that proteins extracted from old rats are more hydrophobic than proteins from young rats. When protein obtained from rat liver extracts was exposed to a metal-catalyzed oxidation (MCO) system comprised of iron/ascorbate/oxygen/hydrogen peroxide, there was a transient increase in the level of hydrophobicity as measured by ANS binding, but after 5 hours of incubation the level returned to that of untreated controls. In contrast, treatment of rat liver protein with 2,2'-azobis(2-amidinopropane) (AAPH), a compound that decomposes in water to form carbon-centered alkyl radicals, there was a progressive increase in hydrophobicity with time, and after 5 hours 30% of the protein was precipitated. This was accompanied by the loss of 5 proteins. Two of these were recovered in the precipitated protein. One of the precipitated proteins was identified as glutathione-S- transferase. These results indicate that there is an age-related increase in the hydrophobicity of proteins in rat liver and that this could be due in part to modification of the protein by free radicals.